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The invention relates to mammalian sphingosine-1-phosphate phosphatase, polynucleotides encoding the enzyme and assays that measure the catabolism of sphingosine-1-phosphate by mammalian sphingosine-1-phosphate phosphatase.
Sphingosine-1-phosphate (SPP) is a bioactive sphingolipid metabolite which regulates diverse biological processes (reviewed in (Goetzl, et al., (1998) FASEB J. 12, 1589-1598 and Spiegel, S. (1999) J. Leukoc. Biol. 65, 341-344.) Many of its actions are reported to be mediated by a family of specific cell surface G-protein coupled receptors (GPCR), known as EDG (endothelial differentiation genes) receptors. Binding of SPP to EDG-1 expressed on endothelial cells reportedly enhances survival (Hisano, et al., (1999) Blood 93, 4293-4299), chemotaxis and in vitro angiogenesis (Wang, et al., (1999) J. Biol. Chem. 274, 35343-35350) and adherens junction assembly leading to morphogenetic differentiation (Lee, et al., (1999) Cell 99, 301-312), whereas binding of SPP to EDG-5 and EDG-3 is reported to induce neurite retraction and soma rounding (Postma, et al., (1996) EMBO J. 15, 2388-2392 and Van Brocklyn, et al., (1999) J. Biol. Chem. 274, 4626-4632). Additional research indicates that SPP induces activation of Gi-gated inward rectifying K+-channels in atrial myocytes (van Koppen, et al., (1996) J. Biol. Chem. 271, 2082-2087) and inhibits motility of melanoma cells (Yamamura, et al., (1997) Biochemistry 36, 10751-10759) through as yet uncharacterized GPCRs.
SPP is also described as performing important roles inside cells. In response to diverse external stimuli, sphingosine kinase, the enzyme that catalyzes the phosphorylation of sphingosine to SPP, is activated (Olivera, et al., (1993) Nature 365, 557-560; Choi, et al., (1996) Nature 380, 634-636; Melendez, et al., (1998) J. Biol. Chem. 273, 9393-9402; Xia, et al., (1998) Proc. Natl. Acad. Sci. USA 95, 14196-14201; Kleuser, et al., (1998) Cancer Res. 58, 1817-1824 and Meyer zu Heringdorf, et al., (1998) EMBO J. 17, 2830-2837). Intracellular SPP in turn mobilizes calcium from internal stores independently of InsP3 (Meyer zu Heringdorf, et al., (1998) EMBO J. 17, 2830-2837 and Mattie, et al., (1994) J. Biol. Chem. 269, 3181-3188), as well as eliciting diverse signaling pathways leading to proliferation (Rani, et al., (1997) J. Biol. Chem. 272, 10777-10783 and Van Brocklyn, et al., (1998) J. Cell Biol. 142, 229-240.) and suppression of apoptosis (Cuvillier, et al., (1996) Nature 381, 800-803; Perez, et al., (1997) Nature Med. 3, 1228-1232; Edsall, et al., (1997) J. Neurosci. 17, 6952-6960; Cuvillier, et al., (1998) J. Biol. Chem. 273, 2910-2916).
Because of its dual function as a ligand and second messenger and its pivotal role in cell growth and survival, the synthesis and degradation of SPP is expected to be tightly regulated in a spatial-temporal manner. Until recently, however, little was known of the enzymes involved in SPP metabolism. A previous report described the purification of sphingosine kinase to apparent homogeneity from rat kidney (Olivera, et al., (1998) J. Biol. Chem. 273, 12576-12583). Subsequently the first mammalian sphingosine kinase was cloned from rat and characterized (Kohama, et al., (1998) J. Biol. Chem. 273, 23722-23728). The kinase is described as belonging to a novel, highly conserved gene family (Kohama, et al., (1998) J. Biol. Chem. 273, 23722-23728 and Nagiec, et al., (1998) J. Biol. Chem. 273, 19437-19442). Enforced expression of the sphingosine kinase markedly enhanced the proliferation and survival of cells, substantiating the importance of intracellularly generated SPP in cell fate decisions (Olivera, et al., (1999) J. Cell Biol. 147, 545-548).
SPP can be metabolized by two distinct pathways. In one pathway, SPP is catabolized via a microsomal pyridoxal phosphate-dependent lyase to palmitaldehyde and phosphoethanolamine, which can then be utilized for the biosynthesis of glycerolipids. In a second pathway, SPP is dephosphorylated by specific phosphatases to sphingosine (Spiegel, et al., (1996) FASEB J. 10, 1388-1397).
Genetic manipulation studies in yeast have demonstrated an important role for long-chain phosphorylated sphingoid bases in growth and survival of yeast after nutrient deprivation and heat stress (Mandala, et al., (1998) Proc. Nat. Acad. Sci. USA 95, 150-155; Gottlieb, et al., (1999) Mol. Cell Biol. Res. Commun. 1, 66-71; Mao, et al., (1999) Biochem. J. 342, 667-675 and Skrzypek, et al., (1999) J. Bacteriol. 181, 1134-1140) in a manner which is reminiscent of their effects on mammalian cells. Recently, the yeast genes encoding the lyase and phosphatase enzymes of these two catabolic pathways were identified in S. cerevisiae (Saba, et al. (1997) J. Biol. Chem. 272, 26087-26090; Mandala, et al., (1998) Proc. Nat. Acad. Sci. USA 95, 150-155 and Mao, et al., (1997) J. Biol. Chem. 272, 28690-28694). While the mammalian counterpart of the yeast SPP lyase has recently been identified (Zhou, et al., (1998) Biochem. Biophys. Res. Commun. 242, 502-507), a specific mammalian SPP phosphatase has not been previously reported.
The yeast SPP phosphatases encoded by LBP1 and LBP2 are members of Type 2 lipid phosphate phosphohydrolases, a family of magnesium independent, membrane-bound enzymes that share sequence conservation within three domains that are predicted to be involved in the coordination and hydrolysis of the phosphate moiety (Stukey, et al., (1997) Protein Sci. 6, 469-472). A search of the yeast genome for enzymes containing the three conserved domains revealed the presence of 4 genes encoding putative Type 2 lipid phosphatases. Two of these, DPP1 and LPP1, were shown to encode phosphatases with activity against phosphatidic acid (PA), lysophosphatidic acid (LPA), and diacylglycerol pyrophosphate (DGPP) (Toke, et al., (1998) J. Biol. Chem. 273, 14331-14338 and Toke, et al., (1998) J. Biol. Chem. 273, 3278-3284). In contrast, LBP1 (also known as YSR2 or LCB3) and LBP2 (YSR3), encode phosphatases with remarkable specificity for phosphorylated sphingoid bases and without activity towards glycerolipid substrates (Mandala, et al., (1998) Proc. Nat. Acad. Sci. USA 95, 150-155; Mao, et al., (1997) J. Biol. Chem. 272, 28690-28694 and Skrzypek, et al., (1999) J. Bacteriol. 181, 1134-1140).
The presence of a high affinity SPP phosphatase activity with enzymatic properties similar to yeast SPP phosphatases has been described in crude rat liver and cerebellum extracts (De Ceuster, et al., (1995) Biochem. J. 311, 139-146). Although three isoforms of Type 2 lipid phosphate phosphohydrolases, known as LPP1/PAP2a, LPP3/PAP2b, and LPP2/PAP2c, have been cloned from mammalian cells (reviewed in (Brindley, et al., (1998) J. Biol. Chem. 273, 24281-24284)), these gene products appear to have broad substrate specificity with similar efficiencies against PA, LPA, SPP, ceramide-1-P, and DGPP, when assayed in vitro in lipid/detergent micelles.
The present invention provides polynucleotides encoding a murine sphingosine-1-phosphate phosphatase (mSPP1), recombinant host cells containing mSPP1 polynucleotides, mSPP1 polypeptides, and methods of using the polynucleotides, polypeptides and host cells to conduct assays of sphingosine-1-phosphate phosphatase activity.
Polynucleotides and polypeptides of mammalian mSPP1, an enzyme involved in the catoblism of sphingosine-1-phosphate (SPP) are provided. The recombinant mSPP1 enzyme is catalytically active in the dephosphorylation of SPP. The enzyme is used in in vitro and whole cell assays to screen for compounds that alter the activity of the protein or interact with mSPP1 and, potentially, alter the expression of mSPP1. The invention includes the polynucleotides, proteins encoded by the polynucleotides, host cells expressing the recombinant enzyme and extracts prepared from host cells expressing the recombinant enzyme, probes and primers, and the use of these molecules in assays.
An aspect of this invention is a polynucleotide having a sequence encoding a mSPP1 protein, or a complementary sequence. In a particular embodiment the encoded protein has a sequence corresponding to SEQ ID NO:3. In other embodiments, the encoded protein can be a naturally occurring mutant or polymorphic form of the protein. In preferred embodiments the polynucleotide can be DNA, RNA or a mixture of both, and can be single or double stranded. In particular embodiments, the polynucelotide is comprised of natural, non-natural or modified nucleotides. In some embodiments, the internucleotide linkages are linkages that occur in nature. In other embodiments, the internucleotide linkages can be non-natural linkages or a mixture of natural and non-natural linkages. In a most preferred embodiment, the polynucleotide has the coding sequence contained in sequence SEQ ID NO:1.
An aspect of this invention is a polynucleotide having a sequence of at least about 25 contiguous nucleotides that is specific for a naturally occurring polynucleotide encoding a mSPP1 protein. In particular preferred embodiments, the polynucleotides of this aspect are useful as probes for the specific detection of the presence of a polynucleotide encoding a mSPP1 protein. In other particular embodiments, the polynucleotides of this aspect are useful as primers for use in nucleic acid amplification based assays for the specific detection of the presence of a polynucleotide encoding a mSPP1 protein. In preferred embodiments, the polynucleotides of this aspect can have additional components including, but not limited to, compounds, isotopes, proteins or sequences for the detection of the probe or primer.
An aspect of this invention is an expression vector including a polynucleotide encoding a mSPP1 protein, or a complementary sequence, and regulatory regions. In a particular embodiment the encoded protein has a sequence corresponding to SEQ ID NO:3. In particular embodiments, the vector can have any of a variety of regulatory regions known and used in the art as appropriate for the types of host cells the vector can be used in. In a most preferred embodiment, the vector has regulatory regions appropriate for the expression of the encoded protein in mammalian host cells. In other embodiments, the vector has regulatory regions appropriate for expression of the encoded protein in other eukaryotes, bacteria, yeasts, insect cells, cyanobacteria or actinomycetes. In some preferred embodiments the regulatory regions provide for inducible expression while in other preferred embodiments the regulatory regions provide for constitutive expression. Finally, according to this aspect, the expression vector can be derived from a plasmid, phage, virus or a combination thereof.
An aspect of this invention is host cell comprising an expression vector including a polynucleotide encoding a mSPP1 protein, or a complementary sequence, and regulatory regions. In a particular embodiment the encoded protein has a sequence corresponding to SEQ ID NO:3. In preferred embodiments, the host cell is a eukaryote, yeast, insect cell, gram-positive bacterium, cyanobacterium or actinomycete. In a most preferred embodiment, the host cell is a mammalian cell.
An aspect of this invention is a process for expressing a mSPP1 protein in a host cell. In this aspect a host cell is transformed or transfected with an expression vector including a polynucleotide encoding a mSPP1 protein, or a complementary sequence. According to this aspect, the host cell is cultured under conditions conducive to the expression of the encoded mSPP1 protein. In particular embodiments the expression is inducible or constitutive. In a particular embodiment the encoded protein has a sequence corresponding to SEQ ID NO:3.
Other aspects of this invention include polynucleotides, expression vectors, host cells and process that employ mSPP1b polynucleotides having the sequence of SEQ ID NO:2 or encoding the amino acid sequence of SEQ ID NO:4.
An aspect of this invention is a purified mSPP1 polypeptide having an amino acid sequence of SEQ ID NO:3 or the sequence of a naturally occurring mutant or polymorphic form of the protein such as the mSPP1b protein having the amino acid sequence of SEQ ID NO:4.
An aspect of this invention is a method of determining whether a candidate compound can alter the activity of a mSPP1 polypeptide. According to this aspect a polynucleotide encoding the polypeptide is used to construct an expression vector appropriate for a particular host cell. The host cell is transformed or transfected with the expression vector and cultured under conditions conducive to the expression of the mSPP1 polypeptide. Cells are disrupted and membranes are collected by centrifugation. The membranes, or mSPP1 polypeptide purified from the membranes are contacted with the candidate. Finally, one measures the activity of the mSPP1 polypeptide in the presence of the candidate. If the activity is lower relative to the activity of the protein in the absence of the candidate, then the candidate is a inhibitor of the mSPP1 polypeptide. In preferred embodiments, the polynucleotide encodes a protein having an amino acid sequence of SEQ ID NO:2 or a naturally occurring mutant of polymorphic form thereof. In other preferred embodiments, the polynucleotide has the sequence of SEQ ID NO:1. In particular embodiments, the relative activity of mSPP1 is determined by comparing the activity of the mSPP1 in a host cell. In some embodiments, the host cell is contacted with the candidate and activity of mSPP1 protein is determined by measuring a cell phenotype that is dependent upon mSPP1 function. Finally, according to this aspect the relative activity can determined by comparison to a previously measured or expected activity value for the mSPP1 activity in the host under the conditions. However, in preferred embodiments, the relative activity is determined by measuring the activity of the mSPP1 in a control cell that was not contacted with a candidate compound. In particular embodiments, the host cell is a mammalian cell and the protein inhibited is the mSPP1 produced by the mammalian cell.
By xe2x80x9caboutxe2x80x9d it is meant within 10% to 20% greater or lesser than particularly stated.
As used herein an xe2x80x9cagonistxe2x80x9d is a compound or molecule that interacts with and stimulates an activity of mSPP1.
As used herein an xe2x80x9cantagonistxe2x80x9d is a compound that interacts with mSPP1 and interferes with the interaction of mSPP1 and SPP.
As used herein an xe2x80x9cinhibitorxe2x80x9d is a compound that interacts with and inhibits or prevents mSPP1 from catalyzing the dephosphorylation of SPP by mSPP1.
As used herein a xe2x80x9cmodulatorxe2x80x9d is a compound that interacts with an aspect of cellular biochemistry to effect an increase or decrease in the amount of a polypeptide of mSPP1 present in, at the surface or in the periplasm of a cell, or in the surrounding serum or media. The change in amount of the mSPP1 polypeptide can be mediated by the effect of a modulator on the expression of the protein, e.g., the transcription, translation, post-translational processing, translocation or folding of the protein, or by affecting a component(s) of cellular biochemistry that directly or indirectly participates in the expression of the protein. Alternatively, a modulator can act by accelerating or decelerating the turnover of the protein either by direct interaction with the protein or by interacting with another component(s) of cellular biochemistry which directly or indirectly effects the change.
An aspect of this invention is a transgenic animal useful for the study of the tissue and temporal specific expression or activity of the mSPP1 gene in a non-human animal. The animal is also useful for studying the ability of a variety of compounds to act as agonists, antagonists or inhibitors of mSPP1 activity or expression in vivo or, by providing cells for culture or assays, in vitro. In an embodiment of this aspect of the invention, the animal is used in a method for the preparation of a further animal which lacks a functional endogenous mSPP1 gene. In another embodiment, the animal of this aspect is used in a method to prepare an animal which expresses a non-native mSPP1 gene in the absence of the expression of a endogenous gene. In particular embodiments the non-human animal is a mouse. In further embodiments the non-native mSPP1 gene is a wild-type mSPP1 gene or a mutant mSPP1 gene.
All of the references cited herein are incorporated by reference in their entirety as background material.